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Introduction

The cell is composed of molecules of different chemical nature, whether they are acidic, basic, or neutral, all contribute to the functioning of the cell. Here, we will discuss the histochemical methods used to demonstrate the acidic components of the cell which mainly include nucleic acids and some other molecules like acid phosphatase, hyaluronic acid, and amino acids like aspartate & glutamate.

All these molecules play critical roles inside living organisms. Hence, their histochemical demonstration becomes essential to studying the structural and functional properties of the tissue. For example, nucleic acids act as hereditary material of an organism, so their demonstration becomes essential while studying their structural property, genetic disorder, or signaling process. The high-level application of nucleic acid demonstration involves the study of gene expression in every tissue. Another example includes acid phosphatase, whose high concentration in the tissue infers the presence of any infection, injury, or prostate cancer.

Now, we know why the histochemical demonstration of these compounds is an essential part of the research. But, before the demonstration, the preparation of material is done, which involves the freezing and fixation of the tissue.

Fixation

For the retention of the compound (analyte) at its maximum inside the tissue, fixation should be properly done. Commonly, formaldehyde is used for this process. But sometimes, as in the case of acid phosphatases, 4% formaldehyde with anhydrous acetone is used.^[3][5]

Demonstration of Nucleic Acids

Nucleic acids (DNA & RNA) are hereditary material present in every living organism. Though DNA constitutes the genetic material of almost all organisms, some organisms like viruses have RNA as their genetic material. The structure of these nucleic acids is composed of a ribose sugar, a phosphate group, and a nitrogenous base.

Histochemical Methods to Demonstrate Nucleic acid

There are numerous convenient methods for the demonstration of nucleic acid. The few most important methods include staining with basophilic compounds, affinity probe technique, and feulgen nuclear reaction.^[7]

1. Basophilic staining: The stains that come under the category of basophilic are toluidine blue, methyl green-pyronin, cuprolinic blue, and chromium-gallocyanin.^[3] These chemicals are used to stain the acidic component of the tissue. The detailed procedure to demonstrate nucleic acids with these stains are described below:
   
   1. Toluidine blue staining: This is a polychromatic basic dye which gives different color depending upon its affinity with the tissue component it stains.
      
      1. Principle: This works via the phenomenon of Hematochromasia. It is the property of a stain to give a different color to the tissue when it reacts with its acidic component.
      2. Requirement: Tissue sample, aqueous Toluidine blue (0.1g in 100 ml distilled water), tertiary butyl alcohol, xylene, and slide.
      3. Procedure:
         1. Deparaffinize the slides and wash it under running water.
         2. Put the slides in toluidine blue stain for 8-10 minutes.
         3. Rinse the slides with tertiary butyl alcohol for 5 minutes to remove the extra stain.
         4. Transfer the slides to xylene and mount it in permount.^[6]
      4. Observation: You will observe the DNA in blue-green (or dark blue) and the RNA in violet color.
   2. Methyl green-pyronin Y staining: This stain is composed of two basic dyes, methyl green and pyronin. This staining method is generally used to study the distribution of neuronal cell bodies and nessels cells.
      
      1. Principle: Methyl green has a high affinity for phosphate group of the nucleic acid, so it stains chromatids in the nucleus. Pyronin is mainly used to stain RNA but it can also stain nucleoli.
      2. Requirement: Sample tissue, HCl, acetate buffer (0.1 M, pH 4), methyl green-pyronin Y (0.2% methyl green in 0.2% pyronin Y made in 0.1 M, pH-4 acetate buffer), tertiary butanol and xylene.
      3. Procedure:
         1. Deparaffinize the slides using xylene and dehydrate it by using graded alcohol.
         2. Wash the slides under running distilled water.
         3. Immerse the section in HCl [you can try it by using different concentrations of HCl (0.01-0.1 N HCl)].
         4. Wash the slides with acetate buffer (0.1 M, pH 4)
         5. Put the slides in a solution of methyl green-pyronin Y for 10 minutes at room temperature.
         6. Rinse the slides with tertiary butanol and then pass it through xylene for mounting.^[10]
      4. Observation: You will observe the chromatin/DNA in blue-green to green color. RNA will be observed in pink to red color.

2. Feulgen Nuclear Reaction

This method is specifically used for DNA staining purposes.

1. 1. Principle: This reaction involves two steps:
      1. Treatment with mild acid causes hydrolysis of the purine-N-C1-glycosidic bond. (In this step, the purine bases get separated from the carbohydrate group).
      2. Reaction of fuchsin-sulfurous-acid with the liberated aldehyde groups occur.
   2. Requirement: Tissue sample, fuchsin-sulfurous-acid reagent (mix basic fuchsin and sulfuric acid), HCl, xylene, distilled water, slide, and Coplin jar.
   3. Procedure:
      1. Deparaffinize the sample containing slide with xylene and then leave it in 95% ethyl alcohol for 24 hours. After 24 hours rinse it with water.
      2. Wash the slide with HCl for 1 minute.
      3. Again, hydrolyze the slide with cold normal HCl for 20 minutes at 50 °C.
      4. Rinse the slide again with normal HCl for 1 minute at room temperature.
      5. For 2 hours, stain the slide in fuchsin-sulfurous-acid.
      6. Pass the slide from three Coplin jars containing acid bleaching solution for 10 minutes.
      7. Rinse the slide in distilled water for 5-7 minutes.
      8. Dehydrate with xylene and then mount the slide. (you can use DPX or clarite for mounting).^[8]
   4. Observation: You will observe DNA in reddish-purple color and cytoplasm in green color.

Demonstration of Acid Phosphatase

Acid phosphatases are enzymes found in lysosomes and their function involves hydrolyzing phosphate under acidic conditions. The rise in the concentration of these enzymes infers the presence of infection, injury, or prostate cancer. The demonstration of this enzyme is done by an azo-dye method.^[4]

Azo-dye Method

Principle: In the tissue, acid phosphatases hydrolyze the naphthol acid phosphate which gives a product of naphthol derivative. This derivative, when it reacts (coupling reaction occurs) with diazonium salt, produces a red color which indicates the position of the enzyme.

Requirement: Sample tissue, alpha-naphthyl acid phosphate, Tetrazotized dianisidine (Brentamine Fast Blue B), 0.1 M Acetate Buffer (pH 5.8), HCl, xylene, ethanol, DPX, and slides.

Procedure:

• Deparaffinize the sample tissue and rinse it with alcohol and then wash under distilled water.
• Dip the slide in a solution composed of alpha-naphthyl acid phosphate and Tetrazotized dianisidine (Brentamine Fast Blue B) in acetate buffer (0.1 M, pH 5.8), for 15 minutes.
• Rinse the slide with 1% HCl followed by rinsing with 80% alcohol (this step is done to minimize any brown background discoloration and to remove any extra dye).
• Dehydrate the slides with absolute ethanol, clean in xylene and then mount it in DPX.^[3]

Observation: Your tissue sample will show red-black color when there will be any activity of acid phosphatase in your sample.

Demonstration of Mucosubstances

These substances are composed of two types of molecules, glycoproteins, and proteoglycans. Glycoproteins are branched molecules containing sialic acid and fucose groups. Whereas, proteoglycans are linear molecules containing uronic acid and sulfated groups. In glycoproteins, sialic acid is present at the free end that making the whole structure negatively charged, whereas, in the proteoglycans (for example glycosaminoglycan), the presence of uronic acid and sulfated group at the free ends make it highly acidic. A hyaluronic acid is a non-sulfated group of glycosaminoglycans (GAG). These are components of the extracellular matrix that also give mechanical support to the cell.

These mucosubstances (acidic and non-sulfated/sulfated) can be demonstrated by various methods like: Hale’s colloidal iron method, Periodic-acid-Schiff’s reaction (PAS), Alcian blue, and Metachromatic dyes.

1. Hale’s colloidal iron method

Principle: At very low pH, carboxyl and sulfate-containing substances absorb the colloidal ferric ions. Prussian blue staining reaction then stains the absorbed ferric substance in blue.

Requirement: Tissue section, 12% acetic acid, 2% aqueous potassium ferrocyanide, colloidal iron suspension (make a working colloidal solution by adding colloidal iron suspension in acetic acid, in equal volumes), and Perl’s solution (mix 2% ferrocyanide and 2% HCl in equal volumes).

Procedure:

• Deparaffinize the section and rinse it with distilled water.
• Again, rinse the slides well in the 12% acetic acid.
• Put the section in a working colloidal solution for 15-20 minutes.
• Rinse the section three times with a 12 % acetic acid solution.
• Put the section in Perl’s solution for 20 minutes.
• Wash the section with distilled water to remove the extra solution.
• Counterstain the section with nuclear-fast-red for 1 minute.
• Dehydrate the section, and mount it in DPX.^[1]

Observation: You will observe the acid mucopolysaccharides stained in deep blue color that shows the presence of glycoproteins (example GAGs).

2. Periodic-acid-Schiff Reaction

Principle: The free hydroxyl group is oxidized by periodic acid to aldehyde. When this aldehyde group comes in contact with Schiff’s reagent, it forms a magenta-colored complex.

Requirements: Tissue sample, Periodic acid solution (1 gm periodic acid in 100 ml distilled water), Schiff’s reagent (add 1 gm fuchsin basic in 100 ml boiling distilled water then add 2 gm sodium metabisulfite and 2 ml HCl), and hematoxylin.

Procedure:

• Deparaffinize the section and wash it in distilled water.
• Treat the section with periodic acid for 5 minutes.
• Rinse the section well with distilled water.
• Put the section in Schiff’s reagent for 10-15 minutes.
• Wash the section well under distilled water to remove the extra chemicals.
• Counterstain the section with hematoxylin for 15 seconds.
• Wash the section again with distilled water.
• Rinse the section with alcohol.
• Clean the slide with xylene and mount it.^[2]

Observation: You will observe the section in the magenta color that shows the presence of proteoglycans.

3. Alcian blue

Principle: Alcian blue is a basic dye which mainly stains acidic mucosubstances that are carboxylated and sulfated by forming a salt bridge with them.

Requirement: Tissue sample, 3% acetic acid solution, Alcian blue solution (pH 2.5) [1gm alcian blue in 100 ml of 3% acetic acid solution], Nuclear fast red solution, and xylene.

Procedure:^[9]

• Deparaffinize the tissue and wash it under running distilled water.
• Stain the section of tissue in the alcian blue solution for 30 minutes.
• Rinse the section in distilled water to remove the extra stain.
• Counterstain the section with nuclear fast red for 5 minutes.
• Wash the section again in distilled water for 1 minute.
• Dehydrate the section, clear and mount it for observation.

Observation: Nuclei will be stained in pink to red, mucosubstances in blue color and cytoplasm will be stained in pale pink color.

Application of Histochemical Demonstration of Acidic Compounds

1. Study of the cellular components: Methyl green pyronin aids in the demonstration of RNA that helps to study cellular components in which RNA concentration is significantly high. For example: nerve cells, fibroblast, and osteoblast.^[5]
2. Study of the embryo developmental processes: RNA is considerably high when the embryonic cell undergoes rapid division. So, RNA staining is also used to study its developmental process.
3. Cancer research: In tumors, the RNA concentration is very high which is demonstrated by using histochemical techniques that involve staining with methyl green pyronin. PAS methods are used to study cancers like leukemia of immature RBCs and adenocarcinoma in which mucin is often secreted.^[7]
4. Study of pathological conditions: PAS method is used to study diseases such as glycogen’s storage disease, Paget’s disease of the breast, Whipple’s disease (macrophages are stained), and fungal infections.
5. Barrett’s esophagus: In this disease, there is an abnormal growth of the esophagus and mucin is secreted in a very high amount. This disease is studied by histochemical demonstration of mucin by staining with alcian blue.
6. Study of infectious disease: GAGs are known to have interaction with bacteria and fungi during pathogenesis. This interaction can be studied by histochemical demonstration of GAGs.^[2]

Conclusion

Histochemical reaction with acidic components of the cell mainly involves the vast application of nucleic acid demonstration because of their major functional role (protein synthesis, gene regulation, and developmental process). Other acidic compounds ( for example, acidic enzymes and GAGs) can also be demonstrated, to study variant pathological conditions. Histochemical techniques have been proven as easy and quick methods for various research and biomedical purposes.

References

1. J. C. Wagner, D. E. Munday, And J. S. Harington (1962). Histochemical demonstration of hyaluronic acid in pleural mesotheliomas. The Journal of Pathology and Bacteriology, 84(1), 73 -78. doi:10.1002/path.1700840109.
2. J. C. Thonard & H. W. Scherp (1962). Histochemical demonstration of acid mucopolysaccharides in human gingival epithelial intercellular spaces. Archives of Oral Biology, 7(2), 125–136. doi:10.1016/0003-9969(62)90001-8.
3. J. F. Burton, (1954). Histochemical demonstration of Acid Phosphatase by an improved Azo-dye method. Journal of Histochemistry and Cytochemistry 2(2). doi: https://doi.org/10.1177/2.2.88.
4. M. O. Henneberry, G Engel, J. T. Grayhack (1979). Acid Phosphatase. The Urologic Clinics of North America, 6(3), 629-641. PMID: 388794.
5. N. B. Kurnick, (1955). Histochemistry of Nucleic Acids. International Review of Cytology, 4, 221–268. doi:10.1016/S0074-7696(08)60460-X.
6. Ned Feder, Merrill K. Wolf (1965). Studies nn Nucleic acid Metachromasy. Journal of Cell Biology, 27(2), 327. doi: http://doi.org/10.1083/jcb.27.2.327.
7. R. Korson, (1951). A Differential Stain for Nucleic Acids. Stain Technology, 26(4), 265–270. doi:10.3109/10520295109113222.
8. R. E. Stowell, (1945). Feulgen Reaction for Thymonucleic Acid. Stain Technology, 20(2), 45–58. doi:10.3109/10520294509107130.
9. R. Lev, and S. S. Spicer, (1964). Specific staining of Sulphate Groups with Alcian Blue at low pH. Journal of Histochemistry & Cytochemistry, 12(4), 309–309. doi:10.1177/12.4.309
10. Shoichi Iseki and Tom Mori (1986). Methyl green-pyronin stain distinguishes proliferating from differentiated non proliferating cell nuclei after acid denaturation of DNA. The Journal of Histochemistry and Cytochemistry, 34(5), 683-687. doi: https://doi.org/10.1177/34.5.3701031.